The modular organization of multifunctional peptide synthetases
Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liq...
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| Veröffentlicht in: | Journal of Protein Chemistry 1997-07, Vol.16 (5), p.557-564 |
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| container_title | Journal of Protein Chemistry |
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| creator | Vater, J Stein, T Vollenbroich, D Kruft, V Wittmann-Liebold, B Franke, P Liu, L Zuber, P |
| description | Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed. |
| doi_str_mv | 10.1023/A:1026386100259 |
| format | Article |
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From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.</description><identifier>ISSN: 0277-8033</identifier><identifier>ISSN: 1572-3887</identifier><identifier>EISSN: 1573-4943</identifier><identifier>DOI: 10.1023/A:1026386100259</identifier><identifier>PMID: 9246644</identifier><language>eng</language><publisher>United States: Springer Nature B.V</publisher><subject>Amino Acid Isomerases - chemistry ; Amino Acid Isomerases - physiology ; Amino Acid Sequence ; Amino acids ; Bacterial Proteins ; Binding Sites ; Biosynthesis ; Carrier models ; Chromatography, High Pressure Liquid ; Elongation ; Enzymes ; Gramicidin ; Liquid chromatography ; Liquid phases ; Mass Spectrometry ; Mass spectroscopy ; Modular structures ; Molecular biology ; Molecular Sequence Data ; Multienzyme Complexes - chemistry ; Multienzyme Complexes - physiology ; Mutagenesis ; Peptide Synthases - chemistry ; Peptide Synthases - physiology ; Peptides ; Protein Structure, Secondary ; Reagents ; Structure-Activity Relationship ; Surfactin ; Valine</subject><ispartof>Journal of Protein Chemistry, 1997-07, Vol.16 (5), p.557-564</ispartof><rights>Plenum Publishing Corporation 1997.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c389t-ae14b6be7edde37919ff735fe7a7a16827ecddd3e8aeff679979ea9b9def0a2f3</citedby><cites>FETCH-LOGICAL-c389t-ae14b6be7edde37919ff735fe7a7a16827ecddd3e8aeff679979ea9b9def0a2f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9246644$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vater, J</creatorcontrib><creatorcontrib>Stein, T</creatorcontrib><creatorcontrib>Vollenbroich, D</creatorcontrib><creatorcontrib>Kruft, V</creatorcontrib><creatorcontrib>Wittmann-Liebold, B</creatorcontrib><creatorcontrib>Franke, P</creatorcontrib><creatorcontrib>Liu, L</creatorcontrib><creatorcontrib>Zuber, P</creatorcontrib><title>The modular organization of multifunctional peptide synthetases</title><title>Journal of Protein Chemistry</title><addtitle>J Protein Chem</addtitle><description>Gramicidin S synthetase 2 from B. brevis was affinity labeled at its valine thiolation center with the thiol reagent N-[3H]ethylmaleimide. From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.</description><subject>Amino Acid Isomerases - chemistry</subject><subject>Amino Acid Isomerases - physiology</subject><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Bacterial Proteins</subject><subject>Binding Sites</subject><subject>Biosynthesis</subject><subject>Carrier models</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Elongation</subject><subject>Enzymes</subject><subject>Gramicidin</subject><subject>Liquid chromatography</subject><subject>Liquid phases</subject><subject>Mass Spectrometry</subject><subject>Mass spectroscopy</subject><subject>Modular structures</subject><subject>Molecular biology</subject><subject>Molecular Sequence Data</subject><subject>Multienzyme Complexes - chemistry</subject><subject>Multienzyme Complexes - physiology</subject><subject>Mutagenesis</subject><subject>Peptide Synthases - chemistry</subject><subject>Peptide 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From a tryptic digest of the enzyme-inhibitor complex a radioactive fragment was isolated in pure form by two reversed-phase HPLC steps. It was identified by liquid-phase N-terminal sequencing in combination with electrospray mass spectrometry (ESI-MS) as a hexadecapeptide containing the thiolation motif LGG(H/D)S(L/I). By ESI-MS it was demonstrated that a 4'-phosphopantetheine cofactor was attached to this fragment at its reactive serine. These results are consistent with the "Multiple Carrier Model" of nonribosomal peptide biosynthesis. Site-specific mutagenesis has been performed in thiolation, elongation, and epimerization motifs of some of the modules of surfactin synthetase from B. subtilis to clarify the function of prominent conserved amino acid residues in the intermediate steps of peptide biosynthesis. The modular structure of multifunctional peptide synthetases is discussed.</abstract><cop>United States</cop><pub>Springer Nature B.V</pub><pmid>9246644</pmid><doi>10.1023/A:1026386100259</doi><tpages>8</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0277-8033 |
| ispartof | Journal of Protein Chemistry, 1997-07, Vol.16 (5), p.557-564 |
| issn | 0277-8033 1572-3887 1573-4943 |
| language | eng |
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| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | Amino Acid Isomerases - chemistry Amino Acid Isomerases - physiology Amino Acid Sequence Amino acids Bacterial Proteins Binding Sites Biosynthesis Carrier models Chromatography, High Pressure Liquid Elongation Enzymes Gramicidin Liquid chromatography Liquid phases Mass Spectrometry Mass spectroscopy Modular structures Molecular biology Molecular Sequence Data Multienzyme Complexes - chemistry Multienzyme Complexes - physiology Mutagenesis Peptide Synthases - chemistry Peptide Synthases - physiology Peptides Protein Structure, Secondary Reagents Structure-Activity Relationship Surfactin Valine |
| title | The modular organization of multifunctional peptide synthetases |
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